Transfection of Escherichia coli Spheroplasts III. Facilitation of Transfection and Stabilization of Spheroplasts by Different Basic Polymers

Copyright0 1973 AmericanSocietyforMicrobiology PrintedinU.S.A.

Transfection

of Escherichia coli

Spheroplasts

III.

Facilitation

of Transfection and Stabilization of

Spheroplasts

by

Different Basic

Polymers

WILLIAM D. HENNER, INGRIDKLEBER, AND ROLF BENZINGER

Department of Biology, University ofVirginia, Charlottesville, Virginia22901

Received forpublication30April1973

The only compound which fully replaced protamine sulfate in facilitating transfection of Escherichia coli spheroplasts by phage DNAs was

spermine; poly-L-lysine, poly-L-arginine, DEAE-dextran, histones, and many other polyamines were only slightly effective. Higher-molecular-weight

com-poundswere effective atlower concentrations, andeachcompound had asharp

concentration optimum. The specificity of the facilitation of transfection is discussed in light of Leonard and Cole's (1972) isolation of a polyamine- or

protamine-like, natural competence factor fromStreptococci. By standardizing growth conditions for spheroplast cultures, storing spheroplasts in minimal medium, and adding both protamine sulfate and polyamines to spheroplasts, reproducible competence levels were obtained. Thus, 95% of all spheroplast

preparations gave efficiencies of transfection between 10-3 and 3 x 10-4 for lambda DNA; between 10-6 and 3 x 10-8 forT7DNA; and between 3 x 10-6and

10-7 for T5 phage DNA. The stability of the spheroplastswasextendedfrom 10h

tobetween 2 and 5 days, dependingonthe DNA used fortransfection.

In apreviouspaper(5),protamine sulfatewas

shown to facilitate transfection with seven dif-ferent double-strandedphage DNAsby atleast

300-fold. A majordifficulty with this and other

published Escherichia coli transfection systems

isthathighcompetencelevelsareveryunstable

and that only 1 in 10 spheroplast preparations

giveshigh levels ofcompetence (5).

Other basic polymers were now tested for

their effectson transfection. When polyamines

as well as protamine sulfate were added to

spheroplasts, stable and

highly

competent

prep-arations were

reproducibly

obtained.

MATERIALS AND METHODS

The strains used in thisstudy have been previously described (5). Polymers were purchased from the following sources: heparin, calf thymus histones, arginine-rich histones, lysine-rich histones,and poly-L-lysine (mol wt 3,000-5,000) from Nutritional

Bio-chemicals Corp.; spermine tetrahydrochloride, sper-midine trihydrochloride, putrescine dihydrochloride, and cadaverine dihydrochloride from Calbiochem. 3,3' diaminopropylamine; N',N'-diethyl-1,4-pen-tanediamine; 4-dodecyldiethylene triamine;

tetra-ethylenepentamine; N, N'-bis (2-aminoethyl) 1,3-propanediamine; N,N'-bis (3-aminopropyl)-1,3-propanediamine; 1, 1,4, 7, 7-pentamethyldieth-ylene-triamine; and N, N'-bis [(3-(2 aminoethyla-mine) propyl) ] ethylene diamine from Eastman

Ko-74:

dak. DEAE-dextranwaspurchased fromPharmacia, Inc.; poly-L-ornithine HBr (manufacturer's mol wt

76,000) wasfrom Mann Research Laboratories; and poly-L-lysineHCl (molwt16,400; 75,000; and200,000) andpoly-L-arginineHCl (molwt10,000-20,000)were

fromMiles Laboratories.

Thepurification ofbacteriophage and extraction of phage DNAs has been described previously (5). One unit ofabsorbance at260nmwasassumed to

repre-sent 40,gofdouble-stranded DNA. Standard growth medium for bacterial cultures was the following modification of the formula of Fraser and Jerrel (7):

4.5g ofKH2PO4,8.3 gofNa2HPO4,15g ofCasamino Acids (Difco), 1g ofNH4Cl,10ml ofglycerol,2.5ml of

10%MgSO4,and0.3ml of0.1M CaCl2,perliter. Theoptimalmethoddevelopedforpreparing sphe-roplasts was altered from the previous procedure (5) in the following manner: the absorbance of the ovemight culturewasbetween 8and12intheGilford spectrophotometer (at 550nm, bacteria diluted 10-fold before measurement) when2mlwastransferred

to 400ml of fresh medium. The culture wasshaken vigorously at 37 C. When the absorbance of the culturereached 0.6, the cellswerecentrifuged at room temperature and resuspended in: 2.1 ml of 1.5 M sucrose, followed by 0.6 ml of30% Povite albumin,

0.12ml offreshly dissolvedlysozyme (2 mg/mlin0.25

M Tris buffer, pH 8.1), and 0.24 ml of unbuffered EDTA.

After2min,55mlofminimalPAmedium (100 gof

sucrose, 1 gofglucose, and0.5 gofCasamino Acids [Difco] per literofwater) was added. After 12min at

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roomtemperature without stirring, 1.2 mlofMgSO4 and 0.15 ml of 1% protamine sulfate,aswellas0.1 ml ofafresh solution ofspermine tetrahydrochloride (250

mg/ml in sterile water), were added. Other basic

polymers were also added at thistime; if they were

added to spheroplasts at later times, their effect on

competencewasalways weaker (nomorethan 10%of

the control). Thespheroplastswerestoredoniceand assayed forcompetencewith 1X174 DNAs 3 hlater, sincefullcompetencewasnotdeveloped till that time (5). Such spheroplasts worked best with lambda and

OX174DNAs. For T7 DNAassays,protamine sulfate

wasoften omittedtoobtain highercompetence levels. For T5 assays, spermidine trihydrochloride (250Ag/

ml) or N,N'-bis (3

aminopropyl)-1,3-propanedia-mine (180 jg/ml) was sometimes substituted for

sperminetetrahydrochloride. Forlambda andOX174 DNAassays, spheroplastswerestable for 3to5days.

For 15 assays, theywerestable forno longer than 3

days. For T7DNAassayswithoutprotamine sulfate, spheroplasts were competent between 24 and 72 h afterpreparation.

For the purposes of this paper, facilitation of transfection is definedas anincrease intransfection efficiency at any time after the preparation of the spheroplasts. Stabilization oftransfection is defined

asthe maintenance ofahighlevel of competenceany timebeyond the first10 h.

RESULTS

Table 1 presents the results obtained whena

varietyof basic polymers was usedto facilitate transfection by

OX174

replicative form DNA. None of the compounds was more than 10% as efficient as protamine sulfate in facilitating

transfection, not even the chemically closely

related poly-L-arginine. The optimum

concen-tration of the polymers depended inversely on

the molecular weight; for example,

DEAE-dex-tran (molecular weight 2 x 106) was most

effective at 0.3

mg/ml,

whereas spermine

tet-rahydrochloride (mol wt 348) was best at 400

mg/ml.

Theconcentration optimaobserved in all experiments were relatively sharp (data not

shown), as has also been demonstrated by

Melechen, Hudnik-Plevnik, and Pfeifer(19) for protamine sulfate.

Since some variation in the facilitation of

transfection, dependingon the ageofthe

sphe-roplasts, was observed (Table 1, column 4), transfection assays were performed inthe pres-ence of basic polymers at various times (Table 2). Surprisingly, spermine (which had little or no effect of transfection with 3-h-old

sphero-plasts [seeTable1]) dramaticallyincreasedthe efficiency oftransfection by T7 DNA with 48-and72-h-old spheroplasts when protamine sul-fate had already losteffect. Also, the effect of spermine on

OX174

replicative form

transfec-tion was minimal, showing that facilitation of transfection byspermine depends on theDNA

employed (Table 2). Most fortuitously, adding both protamine sulfate and spermine resulted inthe earlyfacilitation of transfection (whichis observed withprotamine sulfate alone) as well asthestabilization of thespheroplasts (which is observed withspermine alone). Addition of both protamine sulfate andapolyaminewasadopted as the new standard procedure for preparing

spheroplasts (see Materials andMethods). Varioussubstanceswere nowtested in

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combi-nation with protamine sulfate to see if they

TABLE 1. FacilitationofOX174RFtransfectionby basic polymerswithE.coliW3350spheroplasts1to 3h old

Basicpolymera Manufacturer's Average facilita- Range of facili- Optimum No.ofexpt mol wt tionovercontrol tation concn(4g/ml)

Protamine sulfate Heterogeneous 215x 2-1300x 25 18

Filteredprotamine'sulfate 10to50,000 248 x 38-902 x 6 5

Poly-L-arginine 10to20,000 26x 1-145 x 0.5 9

Poly-L-lysine 3 to5,000 20x 3-73x 3 6

Poly-L-ornithine 70,000 19 x 2-52 x 2 4

Histones 7x 1-9x 15 6

Poly-L-lysine 16,400 6 x 4-7x 0.5 4

Poly-L-lysine 75to200,000 4x 2-7x 0.5 5

DEAE-dextran 2,000,000 3x 2-6x 0.3 5

Sperminec 348 2 x 1-4 x 400 5

aAllcompounds wereaddedduringthepreparationofthe spheroplasts (seeMaterials andMethods) since later addition produced much weaker effects.

bProtamine sulfate(Eli Lilly) waspassedsuccessively throughAmiconfilters with molecularweightcutoffs of10,000and 50,000.Thisprocedureremoved 75% of thebiuret-positive material.

cThefollowingcompoundswereineffectiveinfacilitatingtransfection:heparin,spermidinetrihydrochloride,

putrescinedihydrochloride,cadaverinehydrochloride;3,3'-diaminopropylamine;N'N'-diethyl-1, 4-pentanedia-mine;4-dodecyl-diethylene triamine;tetraethylene pentamine;N,N'-bis(2-aminoethyl)-1 3,propanediamine;

N,N'-bis (3-aminopropyl 1,3-propanediamine; 1,1,4,7,7-pentamethyldiethylene triamine; N,N'-bis[(3-(2 aminoethylamine) propy] ethylenediamine.

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TABLE 2. Dependenceoffacilitationoftransfectionontheageof the spheroplasts

Infective centers foundwithspheroplasts containinga Protamine Age ofspheroplasts Ptamne Spermine sulfate

TransfectingDNA when assayed (h) No addition sulfate

(HCIW.

(25gg/ml)

(25Ag/ml) (400jg/ml) _mineplus sper-_(HCI), (400pg/ml)

OX174

RF(5 x

104

molecules) 3 56 1,046 50 1035

4X174 RF(5x 10'molecules) 24 2 18 2 82

4X174RF(5x 10'

molecules)

48 0 7 2 7

T7 DNA(2.6 x 109molecules) 3 0 65 119 82

T7DNA(2.6 x 109molecules) 24 0 14 231 285

T7 DNA(2.6 x 109molecules) 48 1 5 434 1,165

T7 DNA (2.6 x 109molecules) 72 0 0 85

Lambda DNA(8 x 10"molecules) 3 1 2,260 225 2,500

Lambda DNA(8 x 10.molecules) 24 0 1,403 862 3,500

48 5 7,000 4,300 10,000

aAllcompounds were addedduring the preparation of thespheroplastssincelater additionproducedmuch weaker effects.

could exert a

stabilizing

effect on the

sphero-plasts. Table 3 shows that a number of

polya-minesother thanspermine could stabilize

sphe-roplasts fortransfection. The following

conclu-sions are drawn from this table and similar

results from other experiments. (i) Protamine

sulfate gave optimal facilitative effects during

thefirst 10h, as previously noted (5) and older

spheroplasts rapidly lost competence. (ii) For

lambda and 4 X174DNAtransfection, spermine

as well as all ofthe other polyamines listed in

the table

strongly

stabilized

spheroplasts

for 96

h and longer in the presence of protamine

sulfate. (iii) ForT5 and T7 DNA transfection,

only

spermine,

N,N'-bis

(3

aminopropyl)-

1,3-propanediamine, and perhaps spermidine

stabilized

spheroplasts.

Thus the stabilization

of spheroplasts varies considerably depending

onthe DNA usedin the transfection assay.

Table4presents asummaryofrecentworkon

the transfection of E. coli spheroplasts. Four

importantcriteriafor efficient andreproducible

transfection assays are listed: maximum

effi-ciency of transfection, the fraction of

sphero-plastsyielding maximum efficiencies, the

varia-tioninthecompetence ofthe

spheroplasts,

and

the stability of spheroplasts for transfection

assays.

DISCUSSION

The facilitation of transfection by protamine sulfate shows a remarkably high specificity;

only spermine fully substituted for protamine sulfate with some DNAs while 11 other

polya-mines, DEAE-dextran, histones, and polymers

of basic amino acids had little or no effect

(Table 1). Poly-L-arginine, a close chemical

relative of protamine sulfate, was much less

effective thanprotamine sulfate.Inother

trans-fection systems, high specificity has been

ob-served: Koch, Quintrell, and Bishop (12) found

DEAE-dextran andpoly-L-ornithine most

effec-tive in stimulating transfection by poliovirus

RNA, whereas Aoki and Takebe (1) found

poly-L-ornithine effective in facilitating

trans-fection oftobacco

spheroplasts by

tobacco

mo-saic virus RNA; Kohn and Green (13) and

Green (8) showed that spermine enhances

transfection ofBacillus subtilis >a SP82phage

DNA. 8

Each basic polymer had a sharp

concentra-tion optimum for the facilitation of

transfec-tion.

Higher-molecular-weight

compoundswere

effective at lower concentrations (Table 1).

Furthermore,

facilitation of transfection

de-pended on the phage DNA used in the assay

(Table 2). Hopefully, these observations willaid

efforts to facilitate transfection in otherassay

systems.

The

widespread

facilitation of transfection by

basic polymers observed in systems as diverse

asE. coli, mammalian, andtobacco cellsraises

the possibility that protamines or polyamines

are components ofnatural competence factors.

Indeed, Leonard and Cole (16) have isolated a

natural competence factor with polyamine- or protamine-like characteristics from Streptococ-cus challis. Experiments were performed to measure the facilitating effect of protamine

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TABLE 4. Efficiencyof transfection, variations in competence, and stability of competent cells used for E. coli transfection assays

Sphero-plasts

Maximum giving Stability

DNAsfectior

_{transfTyeefccmptenocll}

comfetent

Type

of cell efficiency maximum Variation inefficiencyof ofsphero- References

oftrans- efficiency transfection pat h

fection oftrans- plasts(h)

fection

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plus protamine sulfate

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spheroplasts plus protamine sulfate Lysozyme-EDTA

spheroplasts plus protaminesulfate Lysozyme-EDTA

spheroplasts plus protamine sulfate Lysozyme-EDTA

spheroplasts plus protamine sulfate andpolyamine Lysozyme-EDTA

spheroplasts plus protamine sulfate andpolyamine Lysozyme-EDTA

spheroplasts plus protamine sulfate andpolyamine

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3x 10'

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Ti

Lambda

Lambda 4X174

repli-cative form

Lambda

fd repli-cative form

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T4

T5

P22

T2, T4 T4 Lambda

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VOL.12,1973

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sulfate in the streptococcal transformation sys-tem and of the streptococcal competence factor in the E. coli transfection system. No facilita-tion wasobservedineither case (D. G. Leonard, personal communication; R. Benzinger, unpub-lished observations). The failure of these experi-mentsmight be attributedtothehigh specific-ity of transfection factors observed above and thehigh specificity of streptococcal competence factors (20); they might also be caused by a

fundamental difference between the

transfec-tion and transformation processes (25). Further studies of thebasic proteins in microorganisms should yield interesting results; Kuo and

Au-gust(14) have alreadyshownthatbasicproteins

arearequirement forphage RNA replicase and

that mammalian histones and protamine

sul-fatewill substitute in the reaction.

Previously,ourspheroplastswerestableforat

most 10 h for transfection assays with large phage DNAs, and only 1 outof10preparations gave highcompetence levels (5). The combina-tion ofspermine and protamine sulfate addition

tospheroplastsyieldsstable andhighly

compe-tent spheroplasts (Table 3). This discovery

makes it possible to routinely use the assay in

large-scale experiments and to check the

reproducibilityofresults with the same

sphero-plast preparation.

Spermine may also facilitate transfection as

well as stabilize B. subtilis competent cells (8,

13).This function is in agreement with thedata

of

Taborl.22),

who found that the addition of spermine to spheroplasts greatly stabilized them to osmotic shock. On the other hand,

Razin and

Rozansky

(21) noted that

spermine

alsoexerts atoxic effectonE.coli. Thistoxicity

is considerably reduced for nonmetabolizing

cells, and it is surely no coincidence that our

spheroplasts are most stable in minimal

me-dium, a revival of earlier procedures (3). A

factorwhichhadnotbeen assessedinthisstudy

is the sensitivity ofpolyamines tooxidation

by

air or intracellular enzymes (23); the oxidized

products may be toxic to spheroplasts. When other polyamines were testedfortheir stabiliz-ing effect on spheroplasts, several besides

spermine (seeTable 3) werefoundtobeactive;

fortransfectionby lambda and

OX174

DNAsas manyassixdifferent relativesofsperminewere active; however, for T5 and T7 DNA transfec-tion, mainly N,N'-bis (3

aminepropyl)-1,3-propanediamine and spermidine but not

the other polyamines were effective (Table 3).

These results are also in marked contrast to

those obtained in the facilitation experiments

(Table 1) whereonly sperminecould be

substi-tuted forprotamine sulfate.Thus,facilitationof

transfection and stabilization of spheroplasts may be entirely different processes, especially since somecompounds can only perform one of these two functions. In any case, the beneficial effects ofpolyamines for transfection of E. coli spheroplasts once again point up the impor-tance of polyamines in living systems, as has

been extensively documented in a recent book

(6) and symposium (9).

ACKNOWLEDGMENTS

This investigation wassupported bygrant fromthe

Na-tional Institute for Allergy and Infectious Diseases

(AI-08572), byaPublic Health Research Career Development Award(no.6-K04-GM-50284 GEN) from the National Insti-tute ofGeneral Medical Sciences, and a National Science Foundation subgrantfromthe CenterforAdvancedStudies (NSF GU-1531). We thank Charles Mitchellfortheanalytical ultracentrifugeruns ofallthephageDNAs andC.Leonard for

her generous gift ofStreptococcus challis competence factor andhelpfuldiscussion.

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Escherichia coli spheroplastsinthe transfectionassay

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21. Razin, S., and R. Rozansky. 1959. Mechanism of the

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23. Tabor, H., and C.W.Tabor.1964.Spermidine, spermine, andrelatedamines.Pharmacol. Rev. 16:245-300. 24. Wackernagel, W. 1972. An improved spheroplast assay

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